Galphao is the most abundant G protein in the mammalian brain and mediates neurotransmission by clinically relevent neurotransmitters such as serotonin, dopamine, and opioids. Despite its widespread role, the mechanisms by which Galphao signals is not known. C. elegans Galphao is more than 80% identical to the mammalian protein and acts to inhibit neurotransmitter release by an unknown mechanism. To identify this mechanism, a large-scale genetic screen was conducted for suppressors of an activated G protein-coupled receptor that signals constitutively through Galphao. The mutations isolated from this screen may identify molecules that function downstream of Galphao to inhibit neurotransmitter release. Aim 1 of this proposal is to map and clone the genes identified by the suppressor mutations. One gene has already been cloned, and four others have been mapped to small intervals. Aim 2 proposes using behavioral, cellular, and biochemical analyses to determine whether these genes encode downstream effectors of Galphao and to elucidate the mechanisms by which they inhibit neurotransmitter release. Thus the suppressors will be analyzed for defects in behaviors characteristic of defects in Galphao signaling, such as defects in egg- laying behavior and sensitivity to exogenous serotonin. Several of mutants have already shown such defects. Further experiments will determine in what neurons the genes are expressed and function, thus determining if the genes act in the same cells as Galphao. Aim 3 is based on the identification of one of the suppressor genes as encoding potassium-chloride co-transporter, KCC-3. This has led to the hypothesis that Galphao may inhibit neurotransmitter release by regulating chloride flux across the plasma membrane. This hypothesis will be tested through fluorescent imaging of chloride flux in intact worms using the genetically encoded chloride sensor, Clomeleon, and through genetic analysis of genes that encode proteins involved in chloride flux. The identification and analysis of the molecules that mediate the inhibition of neurotransmitter release by Galphao will advance the current understanding of Galphao signaling, as these mechanisms are currently entirely unknown, and may have broad implications for the understanding of basic nervous system function and the molecular phenomena underlying neuropsychiatric disorders. Public Health: The proposed research will investigate the mechanisms by which specialized signaling proteins known as G proteins function. Many neurotransmitters that are important for the regulation of mood, appetite, sleep, and addiction signal using these proteins. A better understanding of the mechanisms by which G proteins signal may provide a better understanding of how these processes are regulated.